DESCRIPTION(Adapted from applicant's abstract): The principle long-term objective of this project is to provide a detailed biophysical and molecular understanding of exocytotic vesicle fusion and transmitter release in endocrine cells and nerve terminals. Upon electrical stimulation nerve terminals and endocrine cells release a variety of neurotransmitters and neuropeptides by an exocytotic mechanism. This process of neurosecretion is of outstanding importance in a broad range of physiological processes. A detailed understanding of the exocytotic event is thus of substantial interest, in particular for those diseases where neurosecretion is impaired. In exocytosis the vesicles dock at the plasma membrane and fuse with the plasma membrane allowing for release of the vesicle contents through the fusion pore. It is proposed to study individual fusion events and individual docking events using biophysical techniques. Simultaneous measurements of fusion and release will be performed by "patch amperometry", a method which allows simultaneous recording of small capacitance changes and amperometric detection of released transmitter molecules. Experiments in "cell-attached" patched provide information on the requirements of cytosolic conditions and components. To investigate the docking process which is thought to precede fusion in vitro, the applicant will develop a novel approach using an optical trap. They will characterize the physicochemical mechanisms determining docking and transmitter release. These methods will be complemented with biochemical techniques which will be used to characterize the role of specific fusion pore formation (the kiss-and-run mechanism) or alternatively by full fusion followed by conventional endocytosis via coated pits. The long-term aim is to obtain an understanding of the molecular components directly involved in fusion pore formation. Demonstration that the fusion pore in a reconstituted system resembles the fusion poreforming during exocytosis will eventually be of a significance analogous to that demonstrating that specific membrane proteins reconstituted in lipid membranes resemble the properties of specific ion channels.